FetalBrain - Summary

Updated: Tue Nov 4 17:59:00 2014

Introduction

Neurospheres Cortex vs GE

• Genetic mutations to epigenetic regulators and epigenomic alterations are amongst the earliest events in brain cell transformation. PMID: 18772396
• Defining the epigenomic landscape of normal human brain cells is an important first step towards defining the degree of epigenomic deregulation associated with transformation.
• Neurospheres provide a powerful model to study neural stem cells, which are thought to be population from which malignant clones arise.
• Little is known about epigenetic differences that define neurospheres emerged from distinct brain regions.

MZ twins

• The genomes of monozygotic twins are genetically identical but epigenetically distinct providing evidence for the influence of environment on the phenotype.
• When these epigenetic differences arise during development and their consequence is still unknown.

Results

Neurospheres Cortex derived vs GE derived

Asymmetry between Cortex and GE UMRs

• On average, there are , 4.31-fold enrichment in total UMR frequency in Cortex compared to GE, 4.07 in HuFNSC02, and 5.31 in HuFNSC04.
  
• The asymmetry appears to be global, in all chromosomes. It is reproduced in the two individuals, and supported in MeDIP UMRs.
  
• Single CpG level differential methylation is symmetric, but the asymmetry on UMR level can be reproduced with different cutoffs. However, there are no apparent differences in UMR length between Cortex and GE, suggesting that there are more orphan GE UM CpGs that was not able to form UMRs than in Cortex.

Sample	Total.DMR	Hyper.DMR	Hypo.DMR
Cortex-HuFNSC02_GE-HuFNSC02	2178	420	1758
Cortex-HuFNSC04_GE-HuFNSC04	646	91	555

GREAT analysis on Cortex and GE UMRs show brain development terms

• UMRs in both Cortex and GE show enrichment in neuron fate commitment biological process, transcriptional regulation activities, Homeobox protein domain, and abnormal brain development mouse phenotype.
  
• Cortex UMRs are also enriched in forebrain regionalization and pattern specification processes.
  
• GREAT enriched terms are also supported by MeDIP.
  
• In HuFNSC04, Cortex UMRs show abnormal brain development in Mouse Phenotype, but are also enriched in kidney-related processes.

UMR breakdown in chromatin states - TBC

• Pending: ChromHMM

DE genes with proximal UMRs show key factors in brain development

• On average, there are 40 UMRs proximally associated with protein-coding genes (TSS +/- 1500bp), 2.85% of all UMRs.
  
• There are average 8 proximal UMRs associated with DE genes, 19.88% of all proximal UMRs, much lower than observed in breast and supported by MeDIP, maybe more UMRs in enhancers? (ChromHMM). Among them, there are 46.67% unique DE genes change in the same direction as the UMRs, also lower than observed in breast and supported by MeDIP.
  
• The intersect between two individuals are significant in Cortex UMRs. There are 3 DE genes with proximal Cortex UMR that are shared by both individual:
  • GFAP: Glial Fibrillary Acidic Protein. It is used as a marker to distinguish astrocytes from other glial cells during development. Reported associated with many brain disease, including alexanders disease, PMID: 11567214; gliomas, UP reduce tumor and induce differentiation, PMID: 15498217, and astrocytoma, tumor suppressor PMID: 8339269 etc.
    
  • NFIX: Nuclear Factor I/X (CCAAT-Binding Transcription Factor). Also supported by MeDIP. A transcription factor that binds the palindromic sequence in viral and cellular promoters, capable of activating transcription and replication. It is essential for the development of a number of organ systems including brain and bone, e.g. severe neuroanatomical defects (may function in the repression of neural stem cell proliferation or in cell migration) PMID: 18477394, PMID: 19058033.
  • FEZF1: FEZ Family Zinc Finger 1 (ZNF312B). Transcription repressor. Involved in the axonal projection and proper termination of olfactory sensory neurons. Regulates non-cell-autonomously the layer formation of the olfactory bulb development and the interneurons. May be required for correct rostral migration of the interneuron progenitors. DNA demethylation and histone acetylation in its promoter activates its expression and oncogene effect in gastric cancer PMID: 19318583. We observe DN-regulation in Cortex and Cortex UMR though.
    
• There are no intersect in pc genes with proximal GE UMRs between the two individual.
  
• There are no significant DAVID enrichment terms due to the small number of genes.

	proximal.UMRs	unique.genes	DE.DMRs	unique.DE.genes	same.direction
GE_UMRs-HuFNSC02	17	17	4	4	1
Cortex_UMRs-HuFNSC02	53	52	15	14	8
GE_UMRs-HuFNSC04	9	11	3	3	2
Cortex_UMRs-HuFNSC04	82	84	10	9	3

DE genes with proximal Cortex UMRs

• HuFNSC02

name	description	DE
CD58	CD58_molecule	UP
RGS10	regulator_of_G-protein_signaling_10	UP
ADM	adrenomedullin	UP
NKX2-1	NK2_homeobox_1	DN
OTX2	orthodenticle_homeobox_2	DN
USP43	ubiquitin_specific_peptidase_43	DN
GFAP	glial_fibrillary_acidic_protein	UP
NFIX	nuclear_factor_I/X_(CCAAT-binding_transcription_factor)	UP
GAD1	glutamate_decarboxylase_1_(brain,_67kDa)	DN
FZD7	frizzled_family_receptor_7	UP
FZD5	frizzled_family_receptor_5	DN
CXCR7	chemokine_(C-X-C_motif)receptor7	DN
ZAR1	zygote_arrest_1	UP
FEZF1	FEZ_family_zinc_finger_1	DN

• HuFNSC04

name	description	DE
FAM5C	family_with_sequence_similarity_5,memberC	UP
STXBP6	syntaxin_binding_protein_6_(amisyn)	DN
GFAP	glial_fibrillary_acidic_protein	UP
NFIX	nuclear_factor_I/X_(CCAAT-binding_transcription_factor)	UP
MEIS1	Meis_homeobox_1	DN
FSIP2	fibrous_sheath_interacting_protein_2	DN
INSM1	insulinoma-associated_1	DN
FEZF1	FEZ_family_zinc_finger_1	DN
C9orf172	chromosome_9_open_reading_frame_172	DN

DE genes with proximal GE UMRs

• HuFNSC02

name	description	DE
PAX6	paired_box_6	UP
TMEM132B	transmembrane_protein_132B	UP
PID1	phosphotyrosine_interaction_domain_containing_1	DN
ZIC3	Zic_family_member_3	UP

• HuFNSC04

name	description	DE
MN1	meningioma_(disrupted_in_balanced_translocation)_1	DN
PNCK	pregnancy_up-regulated_non-ubiquitously_expressed_CaM_kinase	UP

UMR distal associated genes - TBC

• PENDING

Asymmetry in overlapping UMRs with TFBSs

• Overlap UMRs with transcription factor binding sites and count No. of overlapping TFBSs for each TF showed similar asymmetry between Cortex and GE in both individuals, with TFBSs enriched in Cortex UMRs for most TFs. Opposite trend in HuFNSC01 MeDIP.
  
• However, in general, the correlation of TFBS Cortex UMR vs GE UMR fold change between the two individual is quite low, only 0.2. Same in MeDIP.
  
• There are 15 TFs that are at least 3-fold enriched in TFBSs overlapping Cortex UMRs compared to GE UMRs as shown below.

TF	Cortex02UMR	GE02UMR	Ratio02	Cortex04UMR	GE04UMR	Ratio04
TAF7	12	4	3.00	10	1	10.00
TFAP2A	30	10	3.00	20	3	6.67
USF1	67	21	3.19	31	6	5.17
ELF1	45	14	3.21	39	5	7.80
GATA2	116	36	3.22	43	4	10.75
ESR1	55	17	3.24	9	3	3.00
CTCF	197	56	3.52	105	16	6.56
TFAP2C	32	9	3.56	23	5	4.60
MYC	133	36	3.69	84	5	16.80
FOXA1	89	24	3.71	26	5	5.20
TCF7L2	82	22	3.73	47	4	11.75
ZNF263	31	8	3.88	23	5	4.60
E2F1	35	9	3.89	38	5	7.60
TAL1	16	4	4.00	5	1	5.00
GATA3	63	8	7.88	18	1	18.00

DE genes between Cortex and GE are enriched in neuron development and cell migration

• On average, there are 860 genes differentially expressed between cortex and GE, among them, 454 are upregulated in cortex, and 406 are downregulated.
  
• 382 Cortex up-regulated genes, and 456 GE up-regulated genes are shared by at least two individuals.
  
• DAVID enrichment analysis show significant enrichment in neuronal development and cell migration terms, GE up-regulated genes are enriched in EGF-related protein domains as well.
• There are 22 Cortex up-regulated genes, and 26 GE up-regulated genes shared by all four individuals.
  
• Brain / neuron development related Cortex up-regulated genes (shared by all):
  • SLC1A6: Solute Carrier Family 1 Member 6. Glutamate transporter in cerebellar Purkinje neurons, related to traumatic brain injury.
    
  • PCDH20: Protocadherin 20. Although its specific function is undetermined, the cadherin-related neuronal receptor is thought to play a role in the establishment and function of specific cell-cell connections in the brain.
    
  • GFAP: see above.
    
  • ZIC5: Zic Family Member 5. Essential for neural crest development, converting cells from an epidermal fate to a neural crest cell fate, associated with meningiomas PMID: 20199689.
  • ZIC2: Zic Family Member 2. Related to structural anomaly of the forebrain, and neural tube defects.
    
  • NEFM: Neurofilament, Medium Polypeptide. Neurofilaments comprise the axoskeleton and functionally maintain neuronal caliber. They may also play a role in intracellular transport to axons and dendrites. This protein is commonly used as a biomarker of neuronal damage.
    
  • UNC5C: Unc-5 Homolog C. Receptor for netrin required for axon guidance. Mediates axon repulsion of neuronal growth cones in the developing nervous system upon ligand binding.
    
  • ADAM19: ADAM Metallopeptidase Domain 19. Involved in neurogenesis, cell migration, cell adhesion, cell-cell and cell-matrix interactions, serves as a marker for dendritic cell differentiation. UP in primary brain tumors PMID: 16772875.
    
• Brain / neuron development related GE up-regulated genes (shared by all):
  • VAX1: Ventral Anterior Homeobox 1. Transcription factor that may function in dorsoventral specification of the forebrain. Required for axon guidance and major tract formation in the developing forebrain.
    
  • NRXN3: Neurexin 3. Neuronal cell surface protein that may be involved in cell recognition and cell adhesion.
    
  • FEZF1: see above.
    
  • SIX3: SIX Homeobox 3. Plays a role in eye development.
    
  • ODZ1: Odz, Odd Oz/Ten-M Homolog 1. Involved in neural development, regulating the establishment of proper connectivity within the nervous system.
    
  • EPHA3: EPH Receptor A3. Involved in short-range contact-mediated axon guidance. UP in glioblastoma PMID: 23410976.
    
  • OTX2: Orthodenticle Homeobox 2. Plays a role in the development of the brain and the sense organs, also influences the proliferation and differentiation of dopaminergic neuronal progenitor cells during mitosis. UP in medulloblastomas PMID: 20028867.
    
  • ASTN1: Astrotactin 1. A neuronal adhesion molecule required for glial-guided migration of young postmitotic neuroblasts in cortical regions of developing brain, including cerebrum, hippocampus, cerebellum, and olfactory bulb (Fink et al., 1995).

	UP	DN	DE
HuFNSC01	403	508	911
HuFNSC02	588	640	1228
HuFNSC03	447	227	674
HuFNSC04	378	249	627

DE	name	description
UP	SLC1A6	solute_carrier_family_1_(high_affinity_aspartate/glutamate_transporter),member6
UP	PCDH20	protocadherin_20
UP	C9orf64	chromosome_9_open_reading_frame_64
UP	GFAP	glial_fibrillary_acidic_protein
UP	ZIC5	Zic_family_member_5
UP	NEFM	neurofilament,mediumpolypeptide
UP	QPCT	glutaminyl-peptide_cyclotransferase
UP	UNC5C	unc-5_homolog_C_(C._elegans)
UP	ZIC2	Zic_family_member_2
UP	XKR8	XK,Kellblood_group_complex_subunit-related_family,member8
UP	PYGL	phosphorylase,glycogen,liver
UP	SERINC2	serine_incorporator_2
UP	KIAA1239	KIAA1239
UP	B3GALT1	UDP-Gal:betaGlcNAc_beta_1,3-galactosyltransferase,polypeptide1
UP	CCDC48	coiled-coil_domain_containing_48
UP	NOS1AP	nitric_oxide_synthase_1_(neuronal)adaptorprotein
UP	ADAM19	ADAM_metallopeptidase_domain_19
UP	H1F0	H1_histone_family,member0
UP	ZIC3	Zic_family_member_3
UP	NT5E	5’-nucleotidase,ecto(CD73)
UP	SYNM	synemin,intermediatefilament_protein
UP	C1orf226	chromosome_1_open_reading_frame_226

DE	name	description
DN	DPPA4	developmental_pluripotency_associated_4
DN	VAX1	ventral_anterior_homeobox_1
DN	SHISA6	shisa_homolog_6_(Xenopus_laevis)
DN	NRXN3	neurexin_3
DN	FGFR2	fibroblast_growth_factor_receptor_2
DN	ST8SIA2	ST8_alpha-N-acetyl-neuraminide_alpha-2,8-sialyltransferase_2
DN	SLC32A1	solute_carrier_family_32_(GABA_vesicular_transporter),member1
DN	FEZF1	FEZ_family_zinc_finger_1
DN	AJAP1	adherens_junctions_associated_protein_1
DN	ZNF703	zinc_finger_protein_703
DN	VSIG10L	V-set_and_immunoglobulin_domain_containing_10_like
DN	SIX3	SIX_homeobox_3
DN	SORL1	sortilin-related_receptor,L(DLRclass)Arepeats_containing
DN	LMO1	LIM_domain_only_1_(rhombotin_1)
DN	ODZ1	odz,oddOz/ten-m_homolog_1_(Drosophila)
DN	EPHA3	EPH_receptor_A3
DN	TIMP3	TIMP_metallopeptidase_inhibitor_3
DN	CAMK2N1	calcium/calmodulin-dependent_protein_kinase_II_inhibitor_1
DN	EEF1A2	eukaryotic_translation_elongation_factor_1_alpha_2
DN	OTX2	orthodenticle_homeobox_2
DN	CHL1	cell_adhesion_molecule_with_homology_to_L1CAM_(close_homolog_of_L1)
DN	PCSK1N	proprotein_convertase_subtilisin/kexin_type_1_inhibitor
DN	LMO2	LIM_domain_only_2_(rhombotin-like_1)
DN	ASTN1	astrotactin_1
DN	BASP1	brain_abundant,membraneattached_signal_protein_1
DN	ADCYAP1R1	adenylate_cyclase_activating_polypeptide_1_(pituitary)receptortype_I

Isoforms between Cortex and GE are enriched in cell signaling proteins

• On average, 2054 genes are identified as isoforms between cortex and GE in each individual. 2352 genes are shared in at least two individuals.
  
• Individual specific isoforms between cortex and GE have no significantly enriched terms, suggesting they are more likely random events without biological functions.
  
• Isoforms shared by at least two individuals are enriched in terms related to cellular signaling. InterPro shows enrichment in EGF protein domain.

	DE_genes	DE_exons	with_expressed_genes	isoform_exons	exclude_DE_genes	isoform_genes
cortex01_GE01_summary	911	32372	18968	8440	7962	2447
cortex02_GE02_summary	1228	35196	21880	8163	7374	2298
cortex03_GE03_summary	674	29617	13746	6401	6022	2086
cortex04_GE04_summary	627	22386	11253	4323	4259	1386

Intron retention between Cortex and GE - TBC

• PENDING

mCpG is a stable mark for exon transcription during development

• mCpG at cassette exon boundaries has similar pattern to expressed in both exons.
  
• mCpG for cassette exons between cortex and GE neurospheres shows no significant differences.
  
• Results from both WGBS (HuFNSC02 & HuFNSC04) and MeDIP (HuFNSC01 & HuFNSC02) support the assumption that mCpG is a stable mark for exon transcription during development.
  
• mCpG exon marking is established between neurospheres. Needs further validation against H1.

H3K36me3 in exon bodies - normalization issue?

• H3K36me3 in expressed in both / not expressed exons shows no significant differences between HuFNSC01 and HuFNSC02.
  
• H3K36me3 in cassette exons in HuFNSC01 are enriched in GE compared to cortex. However, it is not reproduced in HuFNSC02, where there is no significant differences between cortex and GE. Not sure what to make of this. Are there any potential bias?

MZ twins

UMR asymmetry between MZ twins in Brain and Cortex

• There is an asymmetry between UMRs in HuFNSC01 and HuFNSC02 in the Brain and Cortex neurosphere, but not in GE neurosphere. Fold change in UMR frequency HuFNSC02/HuFNSC01 in Brain is 2.41, in Cortex is 1.83, and in GE is 0.899.

Sample	Total.DMR	Hyper.DMR	Hypo.DMR
Brain-HuFNSC01_Brain-HuFNSC02	4472	2750	1722
Cortex-HuFNSC01_Cortex-HuFNSC02	3161	1758	1403
GE-HuFNSC01_GE-HuFNSC02	2716	1136	1580

GREAT analysis on MZ UMRs are enriched in Homeobox and brain development

• Homeobox protein domain is enriched in all UMR lists.
  
• Brain development related biological processes are enriched in most lists.

UMR breakdown in chromatin states - TBC

• Pending: ChromHMM

MZ UMRs in Brain have less CpGs in CGIs

• Majority of UMR CpGs overlap with genebody, on average 62.59%. And UMR CpGs are 3-fold enriched in promoter regions, with on average 34.29% UMR CpGs are in promoter regions, 39.54% of CpGs in UMRs overlap with CGIs, 5.34-fold than expected by random.
• Brain seems to have less CGI/promoter UMRs.

DE genes with proximal UMRs between MZ are cell type specific

• On average, there are 405 UMRs proximally (TSS +/- 1500bp) associated with protein-coding genes, 11.75% of all UMRs, similar to UMRs between Cortex and GE.
  
• On average, there are 17 proximal UMRs associated with DE genes, 4.24% of all proximal DMRs, much less than UMRs between Cortex and GE (less functional?). Among them, there are 52.53% unique DE genes change in the same direction as the UMRs, similar to UMRs between Cortex and GE.
  
• Proximal UMR genes are mostly cell type specific, but the overlap is still statistically significant.
  
• No overlap between Brain and Cortex for proximal HuFNSC01 UMR DE genes, only one for HuFNSC02, neuropeptide_Y.
  
• HuFNSC01 UMR DE genes associated with brain development:
  • WNT pathway: SFRP2, WNT3.
    
  • EMX2: Empty Spiracles Homeobox 2. Transcription factor. Acts to generate the boundary between the roof and archipallium in the developing brain. May function in combinations with OTX1/2 to specify cell fates in the developing central nervous system.
    
  • MDGA1: MAM Domain-Containing Glycosylphosphatidylinositol Anchor Protein 1. Required for radial migration of cortical neurons in the superficial layer of the neocortex.
    
  • VAX1: see above.
    
  • DLX1: Distal-Less Homeobox 1. Play a role in the control of craniofacial patterning and the differentiation and survival of inhibitory neurons in the forebrain.
    
  • OLIG1: Oligodendrocyte Transcription Factor 1. Promotes formation and maturation of oligodendrocytes, especially within the brain. Associated with human glial brain tumors PMID: 11526205.
    
• HuFNSC02 UMR DE genes associated with brain development:
  • WNT pathway: SFRP1, SFRP2, WNT7A.
    
  • EPHA8: EPH Receptor A8. Plays a role in short-range contact-mediated axonal guidance during development of the mammalian nervous system.
    
  • NRGN: Neurogranin (Protein Kinase C Substrate, RC3). Acts as a “third messenger” substrate of protein kinase C-mediated molecular cascades during synaptic development and remodeling.
    
  • C1QL4: Complement Component 1, Q Subcomponent-Like 4. May regulate the number of excitatory synapses that are formed on hippocampus neurons.
    
  • SNCB: Synuclein, Beta. May play a role in neuronal plasticity.
    
  • SEMA3D: Sema Domain, Immunoglobulin Domain (Ig), Short Basic Domain, Secreted, (Semaphorin) 3D. Induces the collapse and paralysis of neuronal growth cones. Could potentially act as repulsive cues toward specific neuronal populations.
    
  • VGF: May be involved in the regulation of cell-cell interactions or in synatogenesis during the maturation of the nervous system.

	DMRs	unique.genes	DE.DMRs	unique.DE.genes	same.direction
Brain01_Brain02_hyper	432	473	26	26	10
Brain01_Brain02_hypo	367	409	20	20	13
Cortex01_Cortex02_hyper	587	659	30	28	17
Cortex01_Cortex02_hypo	316	342	24	22	11
GE01_GE02_hyper	369	402	1	1	1
GE01_GE02_hypo	361	398	2	2	0

HuFNSC01 UMRs proximal associated DE genes

CellType	name	description	DM	DE
Brain	SAMD11	sterile_alpha_motif_domain_containing_11	hypo	UP
Brain	ITGA8	integrin,alpha8	hypo	UP
Brain	EMX2	empty_spiracles_homeobox_2	hypo	DN
Brain	ARNTL2	aryl_hydrocarbon_receptor_nuclear_translocator-like_2	hypo	UP
Brain	CPNE8	copine_VIII	hypo	DN
Brain	PCDH17	protocadherin_17	hypo	DN
Brain		Putative_3-phosphoinositide-dependent_protein_kinase_2	hypo	UP
Brain	CPNE7	copine_VII	hypo	UP
Brain	PKDREJ	polycystic_kidney_disease_(polycystin)andREJ_homolog_(sperm_receptor_for_egg_jelly_homolog,seaurchin)	hypo	UP
Brain	THRB	thyroid_hormone_receptor,_beta	hypo	UP
Brain	SFRP2	secreted_frizzled-related_protein_2	hypo	DN
Brain	IER3	immediate_early_response_3	hypo	UP
Brain	ZBTB12	zinc_finger_and_BTB_domain_containing_12	hypo	UP
Brain	MDGA1	MAM_domain_containing_glycosylphosphatidylinositol_anchor_1	hypo	DN
Brain	EPHA1	EPH_receptor_A1	hypo	UP
Brain		Protein_kinase-like_protein_SgK196	hypo	UP
Brain	TOX	thymocyte_selection-associated_high_mobility_group_box	hypo	DN
Brain	NCOA2	nuclear_receptor_coactivator_2	hypo	UP
Brain	GPR64	G_protein-coupled_receptor_64	hypo	UP
Brain	TRPC5	transient_receptor_potential_cation_channel,subfamilyC,member5	hypo	DN
Cortex	ELTD1	EGF,latrophilinand_seven_transmembrane_domain_containing_1	hypo	UP
Cortex	CRABP2	cellular_retinoic_acid_binding_protein_2	hypo	DN
Cortex	SPOCK2	sparc/osteonectin,cwcvand_kazal-like_domains_proteoglycan_(testican)_2	hypo	DN
Cortex	VAX1	ventral_anterior_homeobox_1	hypo	UP
Cortex	LRRC10B	leucine_rich_repeat_containing_10B	hypo	DN
Cortex	NDUFA4L2	NADH_dehydrogenase_(ubiquinone)1alpha_subcomplex,4-like2	hypo	DN
Cortex	FOXO1	forkhead_box_O1	hypo	UP
Cortex	C16orf74	chromosome_16_open_reading_frame_74	hypo	DN
Cortex	WNT3	wingless-type_MMTV_integration_site_family,member3	hypo	DN
Cortex	CHST9	carbohydrate_(N-acetylgalactosamine_4-0)sulfotransferase9	hypo	UP
Cortex	CD97	CD97_molecule	hypo	DN
Cortex	RNF144A	ring_finger_protein_144A	hypo	UP
Cortex	DLX1	distal-less_homeobox_1	hypo	UP
Cortex	OLIG1	oligodendrocyte_transcription_factor_1	hypo	UP
Cortex	SCUBE1	signal_peptide,CUBdomain,EGF-like1	hypo	DN
Cortex	GRIA1	glutamate_receptor,ionotropic,AMPA_1	hypo	DN
Cortex	MMD2	monocyte_to_macrophage_differentiation-associated_2	hypo	UP
Cortex	SEMA3E	sema_domain,immunoglobulindomain_(Ig),shortbasic_domain,secreted,(semaphorin)_3E	hypo	DN
Cortex	VIPR2	vasoactive_intestinal_peptide_receptor_2	hypo	DN
Cortex	AP1S2	adaptor-related_protein_complex_1,sigma2_subunit	hypo	DN
Cortex	SH3KBP1	SH3-domain_kinase_binding_protein_1	hypo	UP
GE	FAM5B	family_with_sequence_similarity_5,memberB	hypo	DN
GE	DLL1	delta-like_1_(Drosophila)	hypo	DN

HuFNSC02 UMRs proximal associated DE genes

CellType	name	description	DM	DE
Brain	EPHA8	EPH_receptor_A8	hyper	UP
Brain	IFI6	interferon,alpha-inducibleprotein_6	hyper	DN
Brain	BMP8A	bone_morphogenetic_protein_8a	hyper	UP
Brain	ALX3	ALX_homeobox_3	hyper	UP
Brain	NRGN	neurogranin_(protein_kinase_C_substrate,_RC3)	hyper	UP
Brain	C1QL4	complement_component_1,qsubcomponent-like_4	hyper	UP
Brain	CSRP2	cysteine_and_glycine-rich_protein_2	hyper	DN
Brain	TTYH2	tweety_homolog_2_(Drosophila)	hyper	DN
Brain	DTNB	dystrobrevin,_beta	hyper	UP
Brain	CXCR7	chemokine_(C-X-C_motif)receptor7	hyper	UP
Brain	VHL	von_Hippel-Lindau_tumor_suppressor	hyper	UP
Brain	EPHA6	EPH_receptor_A6	hyper	UP
Brain	CAMK2N2	calcium/calmodulin-dependent_protein_kinase_II_inhibitor_2	hyper	UP
Brain	TBC1D1	TBC1_(tre-2/USP6,BUB2,cdc16)domainfamily,member1	hyper	DN
Brain	NMU	neuromedin_U	hyper	DN
Brain	FSTL5	follistatin-like_5	hyper	DN
Brain	CARTPT	CART_prepropeptide	hyper	DN
Brain	EDIL3	EGF-like_repeats_and_discoidin_I-like_domains_3	hyper	UP
Brain	CCDC90A	coiled-coil_domain_containing_90A	hyper	UP
Brain		LOC401296_proteinUncharacterized_protein	hyper	UP
Brain	NPY	neuropeptide_Y	hyper	UP
Brain	SFRP1	secreted_frizzled-related_protein_1	hyper	DN
Brain	TOX	thymocyte_selection-associated_high_mobility_group_box	hyper	DN
Brain	ENTPD2	ectonucleoside_triphosphate_diphosphohydrolase_2	hyper	UP
Brain	KLHL4	kelch-like_4_(Drosophila)	hyper	UP
Brain	SOX3	SRY_(sex_determining_region_Y)-box_3	hyper	DN
Cortex	DMRTA2	DMRT-like_family_A2	hyper	DN
Cortex	NPR1	natriuretic_peptide_receptor_A/guanylate_cyclase_A_(atrionatriuretic_peptide_receptor_A)	hyper	UP
Cortex	PTPRE	protein_tyrosine_phosphatase,receptortype,_E	hyper	DN
Cortex	PDE3B	phosphodiesterase_3B,_cGMP-inhibited	hyper	UP
Cortex	LDHA	lactate_dehydrogenase_A	hyper	DN
Cortex	GNG3	guanine_nucleotide_binding_protein_(G_protein),gamma3	hyper	DN
Cortex	METTL7B	methyltransferase_like_7B	hyper	DN
Cortex	NME3	non-metastatic_cells_3,proteinexpressed_in	hyper	DN
Cortex	GRIN2A	glutamate_receptor,ionotropic,N-methyl_D-aspartate_2A	hyper	UP
Cortex	NOL3	nucleolar_protein_3_(apoptosis_repressor_with_CARD_domain)	hyper	DN
Cortex	NR1D1	nuclear_receptor_subfamily_1,groupD,member1	hyper	DN
Cortex	IGFBP4	insulin-like_growth_factor_binding_protein_4	hyper	UP
Cortex	TBX2	T-box_2	hyper	UP
Cortex	LBH	limb_bud_and_heart_development_homolog_(mouse)	hyper	DN
Cortex	INSIG2	insulin_induced_gene_2	hyper	DN
Cortex	WNT7A	wingless-type_MMTV_integration_site_family,member7A	hyper	DN
Cortex	SNCA	synuclein,alpha(non_A4_component_of_amyloid_precursor)	hyper	UP
Cortex	NPNT	nephronectin	hyper	DN
Cortex	SFRP2	secreted_frizzled-related_protein_2	hyper	UP
Cortex	PRR16	proline_rich_16	hyper	UP
Cortex	STC2	stanniocalcin_2	hyper	DN
Cortex	SNCB	synuclein,_beta	hyper	DN
Cortex	ITPR3	inositol_1,4,5-trisphosphate_receptor,type3	hyper	UP
Cortex	NPY	neuropeptide_Y	hyper	DN
Cortex	SEMA3D	sema_domain,immunoglobulindomain_(Ig),shortbasic_domain,secreted,(semaphorin)_3D	hyper	UP
Cortex	VGF	VGF_nerve_growth_factor_inducible	hyper	UP
Cortex	CAV1	caveolin_1,caveolaeprotein,_22kDa	hyper	UP
Cortex	SHROOM2	shroom_family_member_2	hyper	UP
GE	DBC1	deleted_in_bladder_cancer_1	hyper	DN

UMR distal associated genes - TBC

• PENDING

Overlapping UMRs with TFBSs show asymmetric in Brain and Cortex

• Overlap UMRs with transcription factor binding sites and count No. of overlapping TFBSs for each TF showed similar asymmetry in Brain and Cortex, but is symmetric in GE. The correlation between Brain and Cortex is also very low, 0.15.
  
• With 2-fold change cutoff, there are 18 TFs enriched in HuFNSC02 in both Brain and Cortex.

TF	Brain.hypo	Brain.hyper	Ratio.Brain	Cortex.hypo	Cortex.hyper	Ratio.Cortex
ESRRA	1	7	0.14	1	5	0.20
TAL1	10	42	0.24	9	34	0.26
HNF4A	15	49	0.31	11	27	0.41
HNF4G	13	41	0.32	10	29	0.34
ZNF217	6	17	0.35	6	14	0.43
EBF1	49	130	0.38	48	98	0.49
FOS	47	123	0.38	43	118	0.36
GATA1	31	73	0.42	32	80	0.40
HSF1	3	7	0.43	2	6	0.33
FOXA2	26	60	0.43	14	36	0.39
GATA3	35	80	0.44	22	52	0.42
GATA2	58	130	0.45	39	96	0.41
STAT3	41	91	0.45	26	87	0.30
CEBPD	22	48	0.46	23	64	0.36
SMC3	71	150	0.47	62	144	0.43
NR3C1	46	95	0.48	39	90	0.43
RXRA	23	47	0.49	16	35	0.46
HDAC6	1	2	0.50	2	11	0.18

DE genes are cell type specific

• On average, there are 470 DE genes across three cells types.
  
• Majority of DE genes are cell type specific, only 98 are shared between any two cell types.
  
• DE genes in Brain is asymmetric, maybe due to cell heterogenity?
  
• There are much fewer DE genes in GE.
  
• DAVID enrichment analysis between MZ twins in brain and cortex show similar GO term in brain development, but there is no significantly enriched terms in GE.

	UP	DN	DE
brain01_brain02	461	181	642
cortex01_cortex02	248	348	596
GE01_GE02	99	74	173

name	description	Brain	Cortex	GE
LMO1	LIM_domain_only_1_(rhombotin_1)	UP	DN	DN
CXCR7	chemokine_(C-X-C_motif)receptor7	UP	UP	DN
SPP1	secreted_phosphoprotein_1	UP	DN	DN
NPY	neuropeptide_Y	UP	DN	DN
COL2A1	collagen,typeII,alpha1	DN	UP	UP
BCL6	B-cell_CLL/lymphoma_6	DN	DN	DN

Isoforms between MZ are enriched in cell signaling in neurospheres and immune response in Brain

• On average, 2617 genes are identified as isoforms between HuFNSC01 and HuFNSC02 in each cell type. 796 genes are shared by all three cell types.
  
• On average, 1724 genes are identified as isoforms between HuFNSC03 and HuFNSC04 in each cell type. 927 genes are shared between two cell types.
  
• Different regions on the Venn diagram have no significantly enriched terms.
  
• Isoforms between HuFNSC01 and HuFNSC02 in neurospheres show similar terms, related to cell signaling, and blood cell development in brain.

	DE_genes	DE_exons	with_expressed_genes	isoform_exons	exclude_DE_genes	isoform_genes
brain01_brain02_summary	642	32138	16302	8980	8542	2902
cortex01_cortex02_summary	596	26983	15554	7618	7445	2454
GE01_GE02_summary	173	23810	12862	7402	7351	2495
cortex03_cortex04_summary	642	26826	12185	5818	5479	1994
GE03_GE04_summary	545	24752	12223	4582	4422	1454

Intron retention in MZ twins - TBC

• PENDING

Methods

DMR identification

WGBS

• Identify DM CpGs
  • methyl_diff one-sided p-value \(\le\) 0.005
    
  • delta fractional methylation \(\ge\) 0.5
    
  • fractional methylation of one sample \(\ge\) 0.75
    
• Collapse DM CpGs into DMRs
  • adjacent DM CpGs have the same DM status;
    
  • distance between adjacent CpGs (size) \(\le\) 300bp;
    
  • No. of CpGs within each DMR \(\ge\) 3.

MeDIP

• DM CpG identification:
  • delta fractional methylation \(\ge\) 0.6
    
  • fractional methylation of one sample \(\ge\) 0.75
    
• Collapse DM CpGs into DMRs:
  • adjacent CpGs have the same DM status;
    
  • distance between adjacent CpGs \(\le\) 300bp;
    
  • No. of CpGs within each DMR \(\ge\) 4.

Differential gene expression with DEfine

• FDR = 0.01
  
• Minimum sum of RPKM (rmin) = 0.005
  
• Minimum sum of coverage (Nmin) = 25

Isoform identification and junction validation

• DEfine on exons: FDR = 0.01
  
• Exon expressed in one sample (\(\ge\) 10% gene RPKM) and not expressed in the other (\(\le\) 1% gene RPKM)
  
• Gene is not DE: DEfine FDR = 0.01
• Gene is expressed in both samples: gene RPKM > 0.01
  
• Validation: For each isoform exon in the previous pairwise comparison
  • Find junctions associated with this exon with enough coverage, i.e. sum of junction coverage of two samples \(\ge\) 1
  • Identify junctions that RPKM change in the same direction as the exon
  • Junction RPKM > 0.1 in one sample and < 0.1 in the other

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